Abstract
Introduction
Oral squamous carcinoma (OSCC) is one of the most challenging global constraint and currently the third most common cancer in India. Malignant cells exhibit anomalous morphological characteristics like increased and abnormal mitosis which might suffice as a prognostic indicator. Skeletal muscles present in close approximation with the oral epithelium are often encountered by the tumor cells of OSCC which is an uncharted territory and might serve as a critical parameter in assessing the outcome of OSCC. Amalgamation of mitotic count and skeletal muscle invasion can put forward cogent paths in discerning the behavior of these lesions.
Material & methods
A total of 60 histopathologically diagnosed cases of well, moderately and poorly-differentiated OSCC cases were obtained from the archives. Hematoxylin and eosin-stained slides were examined for Mitotic count and Skeletal muscle invasion.
Results
There was a statistically significant increase in Mitotic count from well-differentiated to poorly-differentiated OSCC. Despite the statistically insignificant difference there was a distinct rise of skeletal muscle invasion from well-differentiated to poorly-differentiated OSCC.
Conclusion
The rise in mitotic count with increasing grades of OSCC suggests an increase in their proliferation rate. Owing to the marked elevation of skeletal muscle invasion in higher grades of OSCC, we can assume it to be a reliable predictor of aggressiveness and outcome. Further studies with a uniform sample size and site specificity might help in solidifying their role in assessing the prognosis of OSCC.
Keywords: Oral squamous cell carcinoma (OSCC), Mitotic count, Skeletal muscle invasion, Proliferation, Aggressiveness, Prognosis
1. Introduction
Oral squamous cell carcinoma (OSCC) is a global health constraint and the third most common cancer in India. It is highly invasive and is often accredited to tobacco consumption habit.1 Early diagnosis and meticulous knowledge of the etiologic and biological behavior is still a persistent dilemma for clinicians and histopathologists in coping with the increased morbidity and mortality of OSCC. Confirmed diagnosis of OSCC relies on examining its histopathological picture which might contribute in improving the prognosis.2
Multiple histopathological guides have been implemented over the years, yet their efficiency in assessing the prognosis is inadequate.3 One such histopathologic characteristic is the aberration of mitosis which results in a variety of nuclear abnormalities, including micronuclei, nucleation, broken egg appearance, pyknotic nuclei, and increased number of and/or abnormal mitotic figures.4,5 Tumor cells are known to exhibit elevated or abnormal mitosis which is a valuable asset in grading OSCC by depicting increased proliferation.6, 7, 8 The simultaneous increase in proliferation and invasion of tumor cells into surrounding structures is a key step in disease progression further determining prognosis.9 Muscle tissue provides a rich variety of extracellular matrix(ECM) components, topologies and associated tissue structures including vessels and nerves, which jointly provide molecular and physical cues that guide invasion and, likely, reprogram invading tumor cells.9 There is an abundance of skeletal muscles in close approximation with the oral epithelium, which are encountered by the tumor cells. Skeletal muscle invasion is a relatively uncharted territory in assessing the sequelae of OSCC.10 The muscle cells exhibit signs of innate resistance against the spread of tumor cells.11 This barrier can only be breached by highly aggressive tumors thus implying muscle invasion can serve as a crucial judge for prognosis. Moreover, there have been studies of head and neck cancers and lung carcinomas exhibiting signs of skeletal muscle metastasis associated with increased aggressiveness.12,13 Skeletal muscle invasion being an untouched arena can be studied and correlated along with mitotic count which is an already established prime parameter to assess the proliferation rate of OSCC. Therefore, in the present study authors attempted to analyse and correlate the possible histologic determinants of tumor prognosis – mitotic count and skeletal muscle invasion in different grades of OSCC.
2. Material and methods
This was an observational study conducted in the Department of Oral and Maxillofacial Pathology and Microbiology PGIDS, Rohtak. No formula estimating the sample size have been used in this study. Instead histopathologically diagnosed cases of OSCC graded according to the Broder's criteria (1971) in our archives from the year of 2014–2021 were scanned. A total of 60 cases with presence of muscle tissue in the submucosa region were included in our study which consists of well-differentiated squamous cell carcinoma (WDSCC) (n = 32), moderately differentiated squamous cell carcinoma (MDSCC) (n = 23), and poorly differentiated squamous cell carcinoma (PDSCC) (n = 5).4 3–4μm formalin fixed paraffin sections of the same were stained with hematoxylin & eosin. The following criteria were implemented while selecting the samples.
3. Inclusion criteria
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•
Histopathologically diagnosed cases of oral squamous cell carcinoma with visible muscle tissue.
4. Exclusion criteria
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•
As the included cases were an archival study; there is no need of mentioning about patients.
The demographic data regarding their age, gender, site and habit was obtained from the medical records. The patients included in our study were diagnosed by incisional biopsies and referred to higher centers for better treatment facilities limiting our access to excisional biopsies or any information regarding lymph node status and further follow up of the respective cases.
Hematoxylin and eosin-stained sections were evaluated for Mitotic figures which were identified according to the criteria given by Van Diest et al.5 It states:
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1.
The nuclear membrane must be absent indicating the cells have passed the prophase
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2.
Clear, hairy extensions (condensed chromosome) must be present either clotted (beginning metaphase), in a plane (metaphase/anaphase) or in separate clots (telophase).
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3.
Two parallel, clearly separate chromosome clots to be counted individually as if they are separate mitosis.
5. Scoring criteria
Mitotic count was evaluated by counting mitotic figures in ten high power fields (HPF) (40x) from the area with maximum density and were categorized into three groups for achieving normal distribution of data:
0–10 mitotic figures/10 HPF = 1.
11–20 mitotic figures/10 HPF = 2.
>20 mitotic figures/10 HPF = 3.
In order to correlate both our parameters, extent of muscle invasion was also scored. Despite exhaustive search of literature there were studies which only observed presence or absence of muscle invasion but never scored skeletal muscle invasion in OSCC. In our study we have followed the criteria given by Chinn et al. while studying perineural invasion in OSCC.6 Each case was assessed for the presence or absence of muscle invasion. If muscle tissue was seen but there were no signs of invasion, it was considered as “No invasion”. If presence of muscle invasion was noticed, it was scored as:
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1.
Focal – muscle invasion observed in one focus
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2.
Moderate – muscle invasion observed in 2–5 foci
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3.
Severe – muscle invasion observed in >5 foci.
6. Statistical analysis
All the data were analyzed on SPSS version 28.0.0 (IBM SPSS Statistics). The distribution of demographic details like age, gender and site of lesion was observed in different grades of OSCC by Chi-square test. Kruskal Wallis test was used for assessing mitotic figures and Chi-square test was used for skeletal muscle invasion in different grades of OSCC. Further correlation of mitotic count and skeletal muscle invasion was observed in grades of OSCC by ANOVA analysis. Statistical significance of parameters was assessed with a significance threshold P value of 0.05.
7. Results
The observational study was conducted on 60 cases (47 males, 13 females) with an age distribution of 20–95yrs with the mean age of 52.5yrs.
Clinical profile of the cases showed majority in buccal mucosa (n = 26, 43.3%) followed by tongue (n = 13, 31.6%), mandibular alveolus (n = 7, 11.7%), retromolar area (n = 5, 8.3%), lip (n = 4, 6.7%), floor of the mouth (n = 2, 3.3%), vestibule (n = 2, 3.3%) and alveolus (n = 1, 1.7%).
Out of the 49 cases which showed skeletal muscle invasion, 39 (78%) were observed in the age group of above 40 years. Among them severe invasion was shown by 11 (22.4%) whereas 25 (51%) of them showed moderate invasion. The remaining 3 (6%) showed focal invasion. 23/47 (47%) males showed presence of moderate muscle invasion and 11 (22.4%) showed severe muscle invasion. 3/47 (6%) males exhibited focal muscle invasion. On the other hand, out of 12 females, 8 (16.3%) showed moderate muscle invasion, 3 (6%) showed severe muscle invasion and the remaining 1 (2%) case showed focal invasion (Table 1).
Table 1.
Demographic data distribution showing skeletal muscle invasion.
| DEMOGRAPHIC VARIABLES | Focal invasion | Moderate invasion | Severe invasion | |
|---|---|---|---|---|
| AGE | <40yrs | 1 (2%) | 7 (14%) | 3 (6%) |
| >40yrs | 3 (6%) | 25 (51%) | 11 (22.4%) | |
| GENDER | Male | 3 (6%) | 23 (47%) | 11 (22.4%) |
| Female | 1 (2%) | 8 (16.3%) | 3 (6%) | |
| SITE | Buccal mucosa | 3 (6%) | 13 (26.5%) | 3 (6%) |
| Tongue | 4 (8%) | 7 (14%) | ||
| Floor of mouth | 2 (4%) | |||
| Lip | 1 (2%) | 1 (2%) | 3 (6%) | |
| Retromolar area | 3 (6%) | |||
| Alveolus | 6 (12%) | |||
| Vestibule | 2 (4%) | |||
WDSCC – well-differentiated squamous cell carcinoma, MDSCC – moderately-differentiated squamous cell carcinoma, PDSCC – poorly-differentiated squamous cell carcinoma.
Significance threshold P value = 0.05.
P value for skeletal muscle invasion = 0.804 (>0.05).
When skeletal muscle invasion was corroborated along with the site of the lesion, focal invasion was seen only in buccal mucosa (6%) and lip (2%). Maximum sign of moderate muscle invasion was observed in buccal mucosa comprising of 13/49 (16.3%), followed by maxillary and mandibular alveolus with 6 (12%). Tongue had 4 (8%) cases followed by 3 (6%) in retromolar region and 2 (4%) each in floor of mouth and vestibule. Severe muscle invasion was observed highest in tongue 7/49 (14%) followed by lip and buccal mucosa (6% each).
Upon using Chi-square test, the present study data revealed that 11/60 (18.3%) cases did not show any muscle invasion which included 6 (19%) cases of WDSCC and 5 (21.7%) of MDSCC. While comparing muscle invasion among three grades of OSCC statistically non-significant (p value = 0.8) results were obtained. (Table 2). Significant threshold P value for the present study was considered as 0.05. Out of 49/60 (81.4%) cases which showed skeletal muscle invasion, WDSCC showed 3 (9.4%) cases of focal invasion, 17 (53%) cases of moderate invasion and 6 (19%) cases of severe invasion. MDSCC on the other hand showed only 1 (4.3%) case of focal invasion, 11 (47.8%) cases of moderate and 6 (26.1%) cases of severe invasion. Finally, PDSCC showed no cases of focal invasion but 3 (60%) cases of moderate invasion and 2 (40%) cases of severe invasion.
Table 2.
Frequency of skeletal muscle invasion in different grades of OSCC.
| GRADING | NO INVASION | FOCAL INVASION | MODERATE INVASION | SEVERE INVASION | Chi square value | P value |
|---|---|---|---|---|---|---|
| WDSCC | 6(19%) | 3(9.4%) | 17(53%) | 6(19%) | 3.040 | 0.804 |
| MDSCC | 5(21.7%) | 1(4.3%) | 11(47.8%) | 6(26.1%) | ||
| PDSCC | 0 | 0 | 3(60%) | 2(40%) |
WDSCC – well-differentiated squamous cell carcinoma, MDSCC – moderately-differentiated squamous cell carcinoma, PDSCC – poorly-differentiated squamous cell carcinoma.
Significance threshold P value = 0.05.
P value for skeletal muscle invasion = 0.804 (>0.05).
We analyzed the mitotic count in the present study in different grades of OSCC using Kruskal-Wallis test and further correlated it with skeletal muscle invasion using ANOVA analysis (Table 3). The mean mitotic count increased from 1.96 in WDSCC to 2.39 in MDSCC to 2.80 in PDSCC with a collaborative mean of 2.20. The mean standard deviation in mitotic count stands at 0.576 with a statistically significant difference (p value = 0.001). The mean value for skeletal muscle invasion in WDSCC, MDSCC and PDSCC came out to be 1.72, 1.78 and 2.40 respectively with a combined mean of 1.80. The average standard deviation turned out to be 1.005 and showed no statistically significant difference (p value = 0.375). Since we could not avail any follow up information about the respective cases, the corroboration of the parameters with their long-term evaluation could not be achieved.
Table 3.
Correlation of mitotic count and muscle invasion in different grades of OSCC.
| Grade of OSCC | Number of cases | Mean | Standard deviation | P value | |
|---|---|---|---|---|---|
| MITOTIC COUNT | WDSCC | 32 | 1.96 | 0.474 | 0.001 |
| MDSCC | 23 | 2.39 | 0.583 | ||
| PDSCC | 5 | 2.80 | 0.447 | ||
| MUSCLE INVASION | WDSCC | 32 | 1.72 | 0.991 | 0.375 |
| MDSCC | 23 | 1.78 | 1.085 | ||
| PDSCC | 5 | 2.40 | 0.548 |
8. Discussion
Early diagnosis and exact estimation of the prognosis of OSCC can steer us to implement better treatment modalities. Prognostic importance of abnormal and increased mitotic activity in OSCC has already been emphasized in several studies linking it with increasing grade of malignancy.15 Taking this into account, we have evaluated the mitotic figures in the various grades of OSCC (Fig. 1).
Fig. 1.
Hematoxylin and eosin stained section under 10x showing A-tripolar nuclei in PDSCC, B- nuclei in anaphase in MDSCC, C & D-nuclei in metaphase in WDSCC.
Present study results depicted an increase in mitotic count from WDSCC to PDSCC with a statistically significant difference (p value = 0.001) (Table 3). This rise is also observed in studies conducted by Singh P et al. where the mean mitotic index value ranged from 2.0 in WDSCC, 2.5 in MDSCC and 2.9 in PDSCC thus accentuating the high proliferative nature of OSCC cementing the role of Mitotic count as a prognostic determinator.5
Various clinical and histopathological characteristics of the primary tumor have been studied in the past to assess accurate predictive prognostic factors including, tumor size, degree of differentiation, surgical margins, perineural invasion, lymphovascular invasion and lymphocytic infiltrate.16 Vassen L et al. focused on the cross-talk between cells of the tumor microenvironment and the mandibular bone highlighting hypoxia as a instigator for increasing osteoclastogenesis and bone invasion which in turn leads to increased aggressive potential of OSCC.17 Mohan SP et al. suggested prominence of minor salivary gland changes associated with oral epithelial dysplasia and OSCC. Additionally, he pointed out the higher recurrence rate of OSCC with inadequate salivary gland removal.18 Skeletal muscle invasion is an innate feature observed uniformly in OSCC cases, but adequate attention has not been rendered to it. Oral epithelium lies in close proximity to skeletal muscles which are encountered by tumor cells during proliferation in OSCC.10 Doo et al. also stated that skeletal muscle invasion is an occasional feature observed in malignant tumors.19 A wide array of factors is responsible for the resistance of skeletal muscle including muscle motion, mechanical tumor destruction, inhospitable muscle pH and muscle cells remove lactic acid produced by tumor cells, indicating increased aggressiveness of the tumor.4,15 Despite ECM-based interstitial tissues being well conceptualized as invasion substrates for tumors, categorizing invasion of muscle tissue has not received any importance.10,20, 21, 22 Studies conducted on gastric, breast and melanoma cancer suggested certain endogenous cytokine released by muscle tissue which shows inhibitory effect on proliferation of cancer cells.23, 24, 25 This innate resistance present within muscle tissue can only be conquered by the aggressive phenotypes of the tumor.11 Muzamil J et al. reported involvement of muscle indicates increased aggressiveness in small cell lung carcinomas.13 Additional reports by Doo et al. states that muscle metastasis in a malignant tumor shows worse prognosis than a metastasis to any other organ.14
Chandler K et al. measured skeletal muscle invasion and depth of invasion which were correlated to lymph node metastasis and recurrence in OSCC of tongue.26 However, it was also stated that measuring the depth of invasion (DOI) is a tedious process which yields variable results owing to the inter-observer bias and varying cut-off values quoted in different studies. It is less sensitive, ineffective in frozen sections and often overpowers the skeletal muscle invasion in OSCC.14,23 Liao C-T et al. suggested squamous cell carcinoma (SCC) invasion of the extrinsic skeletal muscle of the tongue, as compared to SCC invasion not involving muscle, were associated with 50% increased local recurrence, 93% increased risk of distant metastases and 55% lower disease-free survival after 96 months, without significant differences in the incidence of lymphatic invasion.27 Furthermore, studies have demonstrated association of lymph node metastasis and lower survival rate with skeletal muscle invasion in OSCC using magnetic resonance imaging (MRI) technique.28 Though this strengthens the link between muscle invasion and aggressiveness of OSCC, there has not been any histopathological exploration for the same in any other site of oral cavity except tongue in the literature.
Keeping all this in mind, owing to the laborious evaluation of depth of invasion, in the present study we have attempted using skeletal muscle invasion as a prognostic parameter in OSCC and correlated it with a proven proliferation marker (mitotic count). Owing to the innate resistance possessed by the skeletal muscle to tumor infiltration, the tissues which show increased invasion clearly possess increased aggressiveness. Furthermore, increased mitotic count in these cases act as a supplemental tool to demonstrate the efficiency of the used parameters in determining prognosis. The highest sign of moderate and severe skeletal muscle invasion was focused in males with more than 40yrs of age (Table 1). Gender predilection prevails in development of OSCC mainly due to the increased tobacco consumption among males. Buccal mucosa had the highest amount of moderate muscle invasion whereas tongue had maximum amount of severe muscle invasion which can be justified by the presence of the skeletal muscles in its vicinity. The cases of focal muscle invasion were very limited, mostly in buccal mucosa and one from the lip.
In the studies conducted by Chandler K et al. and Mani C et al. 29.5% and 61% of the total cases showed absence of skeletal muscle invasion in OSCC of tongue and rendered them less aggressive in comparison to those showing muscular invasion.16,21 The present study results revealed that 11/60 (18.3%) cases did not show any muscle invasion, which might justify the reduced aggressiveness of these lesions. In the present study all 5 cases of PDSCC (100%) showed presence of skeletal muscle invasion in comparison to WDSCC (81%) and MDSCC (78.3%), suggesting a higher aggressive potential of PDSCC. In the remaining 49 (81.7%) cases which showed skeletal muscle invasion, the extent of the skeletal muscle invasion by the tumor cells was graded as focal, moderate and severe and observed in different grades of OSCC (Fig. 2).
Fig. 2.
Hematoxylin and eosin stained sections under 40x showing A- WDSCC showing no muscle invasion, B – WDSCC showing muscle invasion, C- MDSCC showing muscle invasion, D- PDSCC showing muscle invasion.
While assessing skeletal muscle invasion in different grades of OSCC we did not obtain a statistically significant difference (p value = 0.804) (Table 2). There was a gradual decline in no invasion and focal invasion cases from WDSCC to PDSCC. Additionally, signs of moderate and severe skeletal muscle invasion were increased from MDSCC to PDSCC. On the contrary, it was observed that some of the cases of WDSCC showed signs of extensive muscle invasion, which could be due to their procurement from lip and tongue with a proximity to the skeletal muscles. A total of 11 cases with no signs of skeletal muscle invasion were associated with a low mitotic count which suggests a lower proliferative potential and thus might exhibit better prognosis. Henceforth we can hypothesize that there was an increase in the degree of skeletal muscle invasion from WDSCC to PDSCC which solidifies its association with aggressiveness of OSCC.
In the present study, we also correlated mitotic count and skeletal muscle invasion among different grades of OSCC (Table 3). There was an increase in the mean mitotic count from WDSCC to PDSCC revealing a statistically significant difference (p value = 0.001). Skeletal muscle invasion also showed a rise in its mean value from WDSCC to PDSCC with a statistically non-significant difference (p value = 0.375). This might owe to the variability in sample size and site. Despite the results showing no statistically significant correlation, there was a distinct rise in mitotic count with increasing rate of skeletal muscle invasion from WDSCC to PDSCC.
Upon exhaustive searching of literature, there has not been any histopathological study of OSCC in sites other than tongue, focusing on grading the skeletal muscle invasion and its further correlation with a known proliferative marker like mitotic count. Present study is the first strive in assessing the aggressive behavior of tumors which could conquer the tumor resistant microenvironment around skeletal muscles owing to their proliferative nature.
The present study results inferred that degree of skeletal muscle invasion increased from WDSCC to PDSCC with a parallel increase in mitotic count which further suggests that OSCC cases without muscle invasion might have lesser aggressive potential and better prognosis in comparison to cases with severe muscle invasion. In conclusion we could hypothesize that skeletal muscle invasion can be used as a prognostic parameter in OSCC which would further impact the decision regarding treatment modalities. More studies including site specificity and uniform sample size from excisional biopsies along with follow up of the subjects are needed to validate present study results.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of competing interest
Authors have no conflict of interest to disclose.
Contributor Information
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